Abstract The evolution of multiple antibiotic-resistant pathogens for which treatment moieties are limited or absent makes it essential to identify exploitable IMMUNE PATHWAYS to improve the efficacy of vaccines against infectious agents. LT-IIb, a type II heat-labile enterotoxin (HLT), is a potent mucosal and systemic adjuvant that augments antigen (Ag)-specific immune responses and elicits protection against pathogenic challenge in several mouse immunization models. Enhancement of Ag-specific immune responses by LT-IIb, a bacterial ADP-ribsosylating toxin, is regulated, in part, by its capacity to induce expression of IL-6, IL-1?, and several other cytokines by immune cells. Current dogma is that the immunomodulatory properties of LT-IIb are mediated by the toxin?s capacity to ADP-ribosylate the GS? regulatory subunit of the trimeric GS??? complex, which constitutively activates adenylate cyclase (AD) in cells, eliciting a dramatic elevation in the intracellular concentration of cAMP, a strong secondary signaling molecule. Preliminary experiments from our lab, however, CONTRADICT that dogmatic model. We show that: (i) forskolin, an agent that strongly activates AD without requiring ADP-ribosylation of GS?, fails to augment Ag-specific immune responses and does not induce IL-6 or IL-1? in immune cells; (ii) pharmacological agents that inhibit AD and the GS trimer have no effect on elaboration of IL-6 by immune cells; and (iii) LT-IIb(E59K/E110K)], an ADP-ribosylase-deficient mutant HLT, neither enhances immune responses to co-administered Ag nor induces production of cytokines in immune cells. Thus, the capacity of LT-IIb to mediate responses in immune cells requires an intact ADP-ribosylase activity, but does not require GS? , AD, or cellular cAMP. Eukaryotic cells express ADP-ribosylases that modify proteins to regulate non-immune pathways. We surmise that LT-IIb is mimicking an endogenous enzyme that modulates responses in immune cells. Our HYPOTHESIS is that the immune responses augmented by LT-IIb are regulated by the toxin?s capacity to ADP-ribosylate one or more proteins operating within an, as yet, undescribed immunoregulatory pathway in B cells, macrophages, and other types of immune cells. To isolate the protein(s) in murine B cells and macrophages that are modified by LT-IIb, we will employ LT-IIb as a MOLECULAR PROBE and a recombinant polypeptide with strong binding for ADP-ribosylated proteins as an affinity agent. Modified proteins obtained from cell lysates using this affinity agent will be identified using a sophisticated, in-house-developed, high-resolution proteomics technology that is NOT BROADLY AVAILABLE and designed to identify proteins in EXCEPTIONALLY LOW CONCENTRATIONS and WITHIN COMPLEX MIXTURES. In a complementary set of experiments, human proteins modified by LT-IIb will be identified using a ?total human proteome? microarray. These EXPLORATORY experiments will open new avenues of research into the roles of ADP-ribosylation in immunomodulation. On a practical level, defining this new regulatory mechanism will facilitate development of new and safer adjuvants for vaccine use.